Homocysteine inhibits potassium channels in human atrial myocytes

1. A large body of evidence indicates that elevated homocysteine (Hcy) levels portend an increased risk for atrial fibrillation. However, little is known about the electrophysiological effects of Hcy on atrial myocytes. The present study was conducted to investigate the direct effects of Hcy on ion channels in human atria. 2. Whole-cell patch-clamp techniques were used to record potassium currents in human atrial cells. 3. In human atrial myocytes, transient outward potassium currents were significantly decreased by 24.8 +/- 5.9 and 38.4 +/- 10.4% in the presence of 50 and 500 micromol/L Hcy, respectively. The ultrarapid delayed rectifier potassium currents were decreased by approximately 30% when exposed to 500 micromol/L Hcy. The inward rectifier potassium currents were increased by approximately 40% in the presence of 500 micromol/L Hcy. 4. The results of the present study indicate that Hcy, an important risk factor for atrial fibrillation, could cause electrophysiological disturbances of potassium currents in human atrial myocytes.     

Plasma membrane hyperpolarization by cyanide in chromaffin cells: role of potassium channels

Exposure of rat pheochromocytoma (PC12) cells to cyanide produces elevation of cytosolic calcium, impaired Na⁺-H⁺ exchange, membrane lipid peroxidation and release of neurotransmitters. Since these observations suggested cyanide alters plasma membrane function, the present study examined the effect of NaCN on the membrane potential of undifferentiated PC12 cells in suspension. In PC12 cells loaded with the voltage sensitive fluorescent dye, bis-oxonol, cyanide (2.5 – 10 mM) elicited an immediate (within seconds), concentration related decrease in fluorescence, indicating hyperpolarization of the plasma membrane. Increasing extracellular K⁺ concentration to 20 mM blocked the effect of cyanide (5 mM), suggesting cyanide increased K⁺ efflux. Pretreatment with quinine blocked the cyanide-induced hyperpolarization, whereas glyburide had little effect, showing the hyperpolarization produced by cyanide was due to activation of Ca²⁺ sensitive K⁺ channels. Removal of Ca²⁺ from the media did not influence cyanide-induced hyperpolarization. However, buffering intracellular Ca²⁺ by loading cells with the Ca²⁺ chelators, Quin II or BAPTA, abolished the cyanide effect, showing cytosolic Ca²⁺ is a key factor. These findings suggest that cyanide mobilizes Ca²⁺ from intracellular stores which leads to hyperpolarization via the activation of Ca²⁺ sensitive K⁺ channels. Received: 7 October 1993/Accepted: 11 January 1994     

Propranolol inhibits the human ether-a-go-go-related gene potassium channels

Propranolol is a noncardioselective beta-adrenergic antagonist that has been recently reported to prolong the QTc interval on the surface electrocardiogram in humans when overdosed [Farhangi, V., Sansone, R.A. (2003). QTc prolongation due to propranolol overdose. Int. J. Psychiatry Med. 33, 201-202.]. To examine the underlying mechanisms for these clinical findings, we studied the effects of propranolol on the human cardiac potassium channels encoded by the ether-a-go-go-related gene (ERG) using the whole cell voltage-clamp technique. We found that propranolol blocked hERG currents in a concentration-dependent manner with an IC50 of 9.9+/-1.3 microM which is relevant to the predicted plasma level of propranolol in this case report. The present study demonstrated that propranolol can inhibit hERG channels. The interaction between propranolol and hERG channels could lead to delayed cardiac repolarization and might be a molecular mechanism for the previously reported QTc prolongation when propranolol is overdosed.     

Curcumin inhibits hERG potassium channels in vitro

Curcumin is reported to exert antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anti-tumor activities. The human ether-a-go-go related gene (hERG) encodes the rapid component of the delayed rectifier K⁺ currents. Inhibition of hERG K⁺ channels leads to cardiac repolarization prolongation, which contributes to either the anti-arrhythmic effects of anti-arrhythmic drugs, or the pro-arrhythmic effects (induction of long QT syndrome) of some drugs not used for anti-arrhythmias. Since curcumin shows multiple beneficial effects and clinical significance, the aim of the present study is to investigate the effect of curcumin on hERG K⁺ channels, elucidating its potential cardiac therapeutic or toxic effects. In whole-cell patch-clamp experiments, we found that curcumin inhibited hERG K⁺ currents in HEK293 cells stably expressing hERG channels in a dose-dependent manner, with IC₅₀ value of 5.55 μM. The deactivation, inactivation and the recovery time from inactivation of hERG channels were significantly changed by acute treatment of 10 μM curcumin. Incubation of 20 μM curcumin for 24 h reduced the HEK293 cell viability. Intravenous injection of maximal amount of curcumin in rabbits (20 mg/animal) did not affect the cardiac repolarization manifested with QTc value. We conclude that curcumin inhibits hERG K⁺ channels in vitro.     

西地那非通过上调电压依赖性钾通道抗低氧刺激的人肺动脉平滑肌细胞增殖

目的探讨西地那非抗低氧刺激的人肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)增殖的机制与电压依赖性钾通道(voltage-dependent potassium channels,Kv)及环鸟甘酸依赖性蛋白激酶(cGMP-dependent protein kinase,PKG)的关系。方法采用MTT法及细胞免疫荧光法等检测细胞增殖情况;应用膜片钳技术记录低氧刺激PASMCs前后及西地那非或KT-5823干预前后的Kv通道电流,运用Kv1.5抗体透析细胞后的Kv通道电流;应用siRNA干扰技术沉默Kv1.5基因。结果低氧下西地那非组的细胞增殖水平较低氧对照组明显降低;西地那非反转了低氧对细胞K +通道电流,尤其Kv1.5通道电流的降低作用;siRNA组的Kv1.5蛋白表达明显减低,沉默Kv1.5通道基因逆转了西地那非抗低氧刺激的细胞增殖作用;PKG抑制剂KT-5823阻断了西地那非抗低氧刺激的细胞增殖作用,并且反转了西地那非对低氧下的Kv通道电流的上调作用。 结论 西地那非可通过上调Kv通道亚型主要是Kv1.5通道,抑制低氧刺激的人PASMCs增殖,且可能与PKG有关。     

Arylamine N-acetyltransferase in human red blood cells.

N-Acetyltransferase activities associated with erythrocytes from 20 individuals have been determined with p-aminobenzoic acid as substrate. A three-fold variation in Vmax is found. The N-acetyltransferase genotype of the individuals has been determined and there is no correlation between the extent of acetylation measured in the individuals' erythrocytes and the inheritance of alleles at the polymorphic NAT locus. Folate is confirmed to be an inhibitor of arylamine N-acetyltransferase activity measured in erythrocytes. The content of folate in erythrocytes of individuals also varies. The individual with the maximum folate content has the minimum N-acetyltransferase activity. The monomorphic N-acetyltransferase gene from individuals spanning the range of N-acetyltransferase activity have been amplified, using the polymerase chain reaction. The pattern of restriction enzyme digestion of the monomorphic N-acetyltransferase gene with a series of eight restriction enzymes is the same for individuals spanning the activity range of arylamine N-acetyltransferase in their erythrocytes.     

Endothelial potassium channels, endothelium-dependent hyperpolarization and the regulation of vascular tone in health and disease

1. The elusive nature of endothelium-derived hyperpolarizing factor (EDHF) has hampered detailed study of the ionic mechanisms that underlie the EDHF hyperpolarization and relaxation. Most studies have relied on a pharmacological approach in which interpretations of results can be confounded by limited specificity of action of the drugs used. Nevertheless, small-, intermediate- and large-conductance Ca2+-activated K+ channels (SKCa, IKCa and BKCa, respectively) have been implicated, with inward rectifier K+ channels (KIR) and Na+/K+-ATPase also suggested by some studies. 2. Endothelium-dependent membrane currents recorded using single-electrode voltage-clamp from electrically short lengths of arterioles in which the smooth muscle and endothelial cells remained in their normal functional relationship have provided useful insights into the mechanisms mediating EDHF. Charybdotoxin (ChTx) or apamin reduced, whereas apamin plus ChTx abolished, the EDHF current. The ChTx- and apamin-sensitive currents both reversed near the expected K+ equilibrium potential, were weakly outwardly rectifying and displayed little, if any, time- or voltage-dependent gating, thus having the biophysical and pharmacological characteristics of IKCa and SKCa channels, respectively. 3. The IKCa and SKCa channels occur in abundance in endothelial cells and their activation results in EDHF-like hyperpolarization of these cells. There is little evidence for a significant number of these channels in healthy, contractile vascular smooth muscle cells. 4. In a number of blood vessels in which EDHF occurs, the endothelial and smooth muscle cells are coupled electrically via myoendothelial gap junctions. In contrast, in the adult rat femoral artery, in which the smooth muscle and endothelial layers are not coupled electrically, EDHF does not occur, even though acetylcholine evokes hyperpolarization in the endothelial cells. 5. In vivo studies indicate that EDHF contributes little to basal conductance of the vasculature, but it contributes appreciably to evoked increases in conductance. 6. Endothelium-derived hyperpolarizing factor responses are diminished in some diseases, including hypertension, pre-eclampsia and some models of diabetes. 7. The most economical explanation for EDHF in vitro and in vivo in small vessels is that it arises from the activation of IKCa and SKCa channels in endothelial cells. The resulting endothelial hyperpolarization spreads via myoendothelial gap junctions to result in the EDHF-attributed hyperpolarization and relaxation of the smooth muscle.     

The antipsychotic drug chlorpromazine inhibits HERG potassium channels

(1) Acquired long QT syndrome (aLQTS) is caused by prolongation of the cardiac action potential because of blockade of cardiac ion channels and delayed repolarization of the heart. Patients with aLQTS carry an increased risk for torsade de pointes arrhythmias and sudden cardiac death. Several antipsychotic drugs may cause aLQTS. Recently, cases of QTc prolongation and torsade de pointes associated with chlorpromazine treatment have been reported. Blockade of human ether-a-go-go-related gene (HERG) potassium channels, which plays a central role in arrhythmogenesis, has previously been reported to occur with chlorpromazine, but information on the mechanism of block is currently not available. We investigated the effects of chlorpromazine on cloned HERG potassium channels to determine the biophysical mechanism of block. (2) HERG channels were heterologously expressed in Xenopus laevis oocytes, and ion currents were measured using the two-microelectrode voltage-clamp technique. (3) Chlorpromazine blocked HERG potassium channels with an IC(50) value of 21.6 micro M and a Hill coefficient of 1.11. (4) Analysis of the voltage dependence of block revealed a reduction of inhibition at positive membrane potentials. (5) Inhibition of HERG channels by chlorpromazine displayed reverse frequency dependence, that is, the amount of block was lower at higher stimulation rates. No marked changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or changes of the inactivation time constant were observed. (6) In conclusion, HERG channels were blocked in the closed and activated states, and unblocking occurred very slowly.     

Methamphetamine inhibits voltage-gated potassium currents in NG108-15 cells: Possible contribution of large-conductance calcium-activated potassium channels

Methamphetamine (MA), a highly abused amphetamine-like psychostimulant, has surged in popularity worldwide in the last decade. Repeated MA exposure has been shown to affect the alternative splice variant expression of large conductance Ca²⁺-activated K⁺ (BK) channels. It remains unclear whether MA affects BK channel activity. The present study investigated the effects of MA on BK channels in NG108-15 mouse neuroblastoma × rat glioma hybrid cells using whole-cell and cell-attached patch clamp techniques. In whole-cell recordings, the macroscopic K⁺ outward currents were inhibited by MA with an EC₅₀ of 146 μM, but not affected by dopamine (DA). It implies that DA is not involved in the effects of MA on K⁺ outward currents. In cell-attached patches, MA significantly decreased BK channel activity. Moreover, MA significantly decreased the BK channel opener NS1619-evoked whole-cell K⁺ outward currents and BK channel activity. Finally, the effect of MA on membrane potential was examined by current-clamp configuration. MA caused membrane depolarization and application of NS1619 returned the depolarized potential to resting value. These findings suggest that MA might act as an inhibitor of BK channels, and thereby increase the neuronal excitability and enhance neurotransmitter release.     

Improved pharmacodynamics of l-asparaginase-loaded in human red blood cells

To evaluate the modification of pharmacodynamic parameters induced by the administration of l-asparaginase loaded into red blood cells, 13 patients received a single dose of l-asparaginase internalised into the carrier. The enzyme was loaded using a reversible lysis-resealing process. The dose per patient ranged from 30 to 200 IU·kg^–1. Considerable heterogeneity occurred between patients: the level of l-asparaginase circulating after 24 h represented 47% of the total injected dose as compared to 74.8% for red blood cells (RBCs). However, the half-life of the enzyme remaining in the circulation was very similar to that of the RBC carrier, i.e. 29 days and 27 days, respectively, compared with 8–24 h for the free enzyme. Sustained elimination of plasma l-asparagine occurred, the duration of which was dependent on the injected dose. A single injection of 30·IU·kg^–1 was sufficient to eliminate plasma l-asparagine over 10 days. With 150–200 IU·kg^–1 the elimination period was extended to 50 days. These data show that the use of RBCs as carriers of l-asparaginase greatly improves the pharmacodynamic parameters of the drug.     

Cyclovirobuxine D inhibits the currents of HERG potassium channels stably expressed in HEK293 cells

Cyclovirobuxine D (CVB-D) has been widely used for treatment of cardiac insufficiency and arrhythmias in China. The antiarrhythmic and proarrhythmic potential of this drug might be concerned with prolongation of action potential duration and QT interval. Human-ether-a-go-go-related gene (HERG) has an important role in the repolarization of the cardiac action potential. This study investigated for the first time the effect of CVB-D on HERG channels stably expressed in HEK293 cells using the whole-cell patch-clamp technique. CVB-D inhibited HERG current (IHERG) in a concentration-dependent manner with an IC50 of 19.7 μM. IHERG blockade required channel activation and was time-dependent, suggesting an open channel block. Moreover, IHERG inhibition by CVB-D was relieved by depolarization to a highly positive membrane potential (+80 mV) that favored HERG channel inactivation. These findings suggested that CVB-D inhibit HERG channels in the open states. CVB-D had no effect on HERG current kinetics. Thus, we conclude that CVB-D inhibits HERG encoded potassium channels and this action might be a molecular mechanism for the previously reported APD prolongation and QT interval prolongation with this drug.     

Methylphenidate, but not other CNS stimulants, inhibits red blood cell calcium-activated potassium efflux

The first-order rate constant of net potassium efflux, measured in human red blood cell (RBC) suspensions by means of a K+-sensitive electrode, was increased 26 fold (from 0.0025 min-1 to 0.0656 min-1) by 0.5 microM of the calcium ionophore A 23187. Both the basal or the calcium-stimulated potassium efflux remained unchanged following the addition of different CNS stimulants (nikethamide (1 mM), pentylenetetrazol (1mM), doxapram (1 mM), strychnine (0.1 mM), picrotoxin (0.1 mM), or nomifensine (0.1 mM). Methylphenidate (10-100 microM), however, inhibited in a concentration-dependent manner the calcium-stimulated, but not the basal potassium efflux. An IC50 of 190 microM was estimated for this effect.     

HPLC法测定人红细胞中的S-腺苷同型半胱氨酸和S-腺苷甲硫氨酸

目的建立HPLC法测定人红细胞中S-腺苷同型半胱氨酸(SAH)和S-腺苷甲硫氨酸(SAM)浓度的方法。方法人红细胞经同体积醋酸盐缓冲液稀释后,经10%高氯酸蛋白沉淀后直接进样。采用Athena C18 WP柱(150 mm×4.6 mm,3μm),柱温35℃,流动相为含5 mmol·L-1庚烷磺酸钠的50 mmol·L-1 Na H2PO4缓冲液(pH 4.0)-甲醇(80:20,v/v),流速:1.0 m L·min-1,检测波长254 nm。采用外标法定量。结果SAH和SAM分别在0.025~10 mg·L-1和0.05~20 mg·L-1内线性关系良好,SAH和SAM的日内、日间RSD均<12%,加样回收率在95.5%~106.3%,样品提取后在24 h内稳定性良好。结论该法快速、简便、准确,适用于SAH和SAM的体内分析检测。     

The fungal neurotoxin lolitrem B inhibits the function of human large conductance calcium-activated potassium channels

The effects of the mycotoxin lolitrem B on the function of hSlo large conductance calcium-activated potassium channels expressed in HEK293 cells have been investigated using inside-out membrane patches. Lolitrem B potently inhibited hSlo potassium currents activated by depolarising voltage pulses in the presence of 10 microM free calcium. At a concentration of 100 nM, lolitrem B rapidly and completely inhibited outward potassium currents. The concentration that produced half-maximal inhibition was 3.7 nM, indicating a high apparent affinity for hSlo channels. This is the first time that a molecular site of action has been identified for a compound of the lolitrem structural class of indole diterpene and identifies a novel BK channel blocker.     

Lipid peroxidation and loss of potassium from red blood cells produced by phototoxic quinolones.

Alterations of the cationic permeability of red blood cell membranes induced by the photosensitiser nalidixic acid were demonstrated by evaluating the potassium loss from intact erythrocytes. The results show that an increase in intracellular potassium efflux, precedes the photohemolysis induced by nalidixic acid. The addition of a nonpermeable osmotic solute, such as sucrose, inhibited photohemolysis but not the potassium loss, indicating a colloid osmotic lysis. Lipid peroxidation induced by nalidixic acid and other photosensitiser quinolones (oxolinic acid and rosoxacin) was time irradiation-dependent. Although rosoxacin was the most photoperoxidative, none of the three quinolones studied produced significant lipid peroxidation. However, of the three quinolones studied, only rosoxacin considerably diminished the percentage of the cholesterol extracted from red blood cell membranes. It is postulated that the increased cation permeability induced by nalidixic and oxolinic acids cannot be attributed to cholesterol oxidation nor to lipid peroxidation; a more probable mechanism is photo-oxidation of amino acid residues of the membrane proteins. However, the lysis induced by rosoxacin is caused by photo-oxidation of cholesterol, not excluding other cellular targets.     

人心肌细胞钾道通研究进展

钾通道是心肌细胞上一类具有重要功能的离子通道,是Ⅲ类抗心律失常药物作用的靶点。近年来,随着分子生物学技术的引入、钾通道克隆的出现及人心肌细胞成功分离和单通道的研究,对人心肌细胞上钾通道的激活和失活动力学特征,电压门控机制及离子的选择性等电生理学特性和药理学意义有了一个较全面深入的了解。这些对于抗心律失常药物的开发与研究具有重要的理论指导意义。     

Orthovanadate inhibits adrenaline-induced hyperpolarization in amphibian sympathetic ganglia

The adrenaline-induced hyperpolarization of neurones in sympathetic ganglia of Rana pipiens was recorded by means of the sucrose gap technique. This hyperpolarization was inhibited in a concentration-dependent manner by sodium orthovanadate (0.1-1.0 mM). Although orthovanadate inhibited the hydrolysis of ATP by preparations of microsomal Na+/K+ ATPase from frog nerve (IC50 = 3.0 microM), extracellularly-applied orthovanadate did not inhibit electrogenic sodium pumping in intact sympathetic ganglia of Rana pipiens. This and other observations that extracellularly-applied orthovanadate did not enter sympathetic neurones and did not have access to intracellular enzyme systems. It is suggested that orthovanadate acts at an extracellular site to inhibit the adrenaline-induced hyperpolarization.     

In vitro uptake of salicylate by human red blood cells

Distribution and binding properties of sodium salicylate to human red blood cells were studied under various experimental conditions. The effect of tonicity and hemolysis on the steady state level of the drug within the human red blood cells were accounted for in this study. When the washed cells were suspended in normal saline solution, the drug was so rapidly permeated into red cells. Since the pH of the system forces nearly complete ionization of the drug, ionic diffusion through aqueous pores is thought to be the mode of salicylate transport. Human red cell binding capacity and association constant for salicylate were estimated. This work supports the view that the red cells act as an important reservior of salicylate.     

Discovery of a retigabine derivative that inhibits KCNQ2 potassium channels

Aim:

Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies.

Methods:

A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System.

Results:

A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC₅₀ value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation.

Conclusion:

The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.